In this project, we focused our studies on the genetic basis both of poxvirus virulence and of host resistance to virus infection. The acquired knowledge should contribute toward development of safe, effective recombinant vaccinia virus vaccines for animal and human use. A newly identified ectromelia virus open reading frame, predicted to encode a 28 kDa protein, was shown to interact with DNA-presumably through a zinc finger motif in the carboxyl terminal region of the protein. The protein was expressed at low levels in the infected cell, and preliminary evidence suggested a nuclear site of localization. A 28 kDa mutant replicated normally in tested tissue culture cell lines but at least 100-fold less efficiently in tissues from the infected mouse, and spread more slowly from the primary site of infection to spleen and liver. The LD50 of the mutant virus was at least 1000-fold lower than the parent virus. This gene appears to be an important poxvirus virulence determinant in the mousepox model. The gene is important for virus replication in the primary peritoneal macrophage of the A strain mouse. Treatment with IFN-gamma allowed mouse macrophages to restrict the replication of ectromelia, vaccinia, and herpes simplex viruses while producing nitric oxide. Inhibitors of nitric oxide synthase blocked the macrophages' antiviral activity. Virus replication was diminished in epithelial cells transfected with cDNA encoding inducible nitric oxide synthase or treated with organic compounds that generate nitric oxide. In mice, an inhibitor of nitric oxide synthase converted sublethal, resolving ectromelia virus infection into fulminant mousepox. Thus, induction of nitric oxide synthase was necessary and sufficient for a substantial antiviral effect of IFN-gamma in vitro and in vivo. Synthetic nitric oxide donor compounds show broad- spectrum antiviral activity in vitro.